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Yorodumi- EMDB-13211: Structure of a DNA-loaded MCM double hexamer engaged with the Dbf... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-13211 | |||||||||||||||
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Title | Structure of a DNA-loaded MCM double hexamer engaged with the Dbf4-dependent kinase | |||||||||||||||
Map data | Auto-refined structure of the DNA-bound MCM double hexamer engaged with DDK | |||||||||||||||
Sample |
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Keywords | Kinase / Mcm2-7 helicase / nucleoprotein complex / DNA replication / REPLICATION | |||||||||||||||
Function / homology | Function and homology information positive regulation of spindle attachment to meiosis I kinetochore / positive regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / positive regulation of DNA replication initiation / positive regulation of kinetochore assembly / positive regulation of meiotic DNA double-strand break formation / negative regulation of exit from mitosis / Dbf4-dependent protein kinase complex / positive regulation of meiosis I / regulation of cell cycle phase transition / positive regulation of nuclear cell cycle DNA replication ...positive regulation of spindle attachment to meiosis I kinetochore / positive regulation of meiotic DNA double-strand break formation involved in reciprocal meiotic recombination / positive regulation of DNA replication initiation / positive regulation of kinetochore assembly / positive regulation of meiotic DNA double-strand break formation / negative regulation of exit from mitosis / Dbf4-dependent protein kinase complex / positive regulation of meiosis I / regulation of cell cycle phase transition / positive regulation of nuclear cell cycle DNA replication / mitotic DNA damage checkpoint signaling / MCM core complex / Assembly of the pre-replicative complex / Switching of origins to a post-replicative state / MCM complex binding / nuclear DNA replication / mitotic DNA replication preinitiation complex assembly / premeiotic DNA replication / pre-replicative complex assembly involved in nuclear cell cycle DNA replication / mitotic DNA replication / Activation of the pre-replicative complex / CMG complex / single-stranded 3'-5' DNA helicase activity / nuclear pre-replicative complex / MCM complex / Activation of ATR in response to replication stress / DNA replication preinitiation complex / protein-containing complex localization / replication fork protection complex / mitotic DNA replication checkpoint signaling / mitotic DNA replication initiation / double-strand break repair via break-induced replication / silent mating-type cassette heterochromatin formation / regulation of DNA-templated DNA replication initiation / single-stranded DNA helicase activity / DNA strand elongation involved in DNA replication / DNA unwinding involved in DNA replication / nuclear replication fork / 3'-5' DNA helicase activity / DNA replication origin binding / chromosome, centromeric region / subtelomeric heterochromatin formation / DNA replication initiation / protein serine/threonine kinase activator activity / helicase activity / chromosome segregation / heterochromatin formation / single-stranded DNA binding / DNA helicase / chromosome, telomeric region / non-specific serine/threonine protein kinase / phosphorylation / positive regulation of protein phosphorylation / cell division / protein serine kinase activity / protein serine/threonine kinase activity / centrosome / DNA damage response / chromatin binding / chromatin / signal transduction / ATP hydrolysis activity / zinc ion binding / nucleoplasm / ATP binding / identical protein binding / nucleus / metal ion binding / cytoplasm Similarity search - Function | |||||||||||||||
Biological species | Saccharomyces cerevisiae S288C (yeast) / Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||||||||
Authors | Greiwe JF / Locke J / Nans A / Costa A | |||||||||||||||
Funding support | European Union, 4 items
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Citation | Journal: Nat Struct Mol Biol / Year: 2022 Title: Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase. Authors: Julia F Greiwe / Thomas C R Miller / Julia Locke / Fabrizio Martino / Steven Howell / Anne Schreiber / Andrea Nans / John F X Diffley / Alessandro Costa / Abstract: Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM ...Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication. | |||||||||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_13211.map.gz | 338 MB | EMDB map data format | |
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Header (meta data) | emd-13211-v30.xml emd-13211.xml | 40.8 KB 40.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_13211_fsc.xml | 16.9 KB | Display | FSC data file |
Images | emd_13211.png | 148.7 KB | ||
Masks | emd_13211_msk_1.map | 421.9 MB | Mask map | |
Filedesc metadata | emd-13211.cif.gz | 11.6 KB | ||
Others | emd_13211_additional_1.map.gz emd_13211_additional_2.map.gz emd_13211_half_map_1.map.gz emd_13211_half_map_2.map.gz | 28.9 MB 3.1 GB 338.6 MB 339.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-13211 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-13211 | HTTPS FTP |
-Validation report
Summary document | emd_13211_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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Full document | emd_13211_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | emd_13211_validation.xml.gz | 24.6 KB | Display | |
Data in CIF | emd_13211_validation.cif.gz | 32.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13211 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-13211 | HTTPS FTP |
-Related structure data
Related structure data | 7p5zMC 7p30C C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_13211.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Auto-refined structure of the DNA-bound MCM double hexamer engaged with DDK | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_13211_msk_1.map | ||||||||||||
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Density Histograms |
-Additional map: Sharpened map
File | emd_13211_additional_1.map | ||||||||||||
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Annotation | Sharpened map | ||||||||||||
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Density Histograms |
-Additional map: LAFTER filtered map, which was used for docking...
File | emd_13211_additional_2.map | ||||||||||||
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Annotation | LAFTER filtered map, which was used for docking of the Dbf4 BRCT domain. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_13211_half_map_1.map | ||||||||||||
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Density Histograms |
-Half map: #2
File | emd_13211_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
+Entire : DNA-loaded MCM double hexamer engaged with the dimeric Dbf4-depen...
+Supramolecule #1: DNA-loaded MCM double hexamer engaged with the dimeric Dbf4-depen...
+Macromolecule #1: DNA replication licensing factor MCM2
+Macromolecule #2: DNA replication licensing factor MCM3
+Macromolecule #3: DNA replication licensing factor MCM4
+Macromolecule #4: Minichromosome maintenance protein 5
+Macromolecule #5: DNA replication licensing factor MCM6
+Macromolecule #6: DNA replication licensing factor MCM7
+Macromolecule #7: Cell division control protein 7
+Macromolecule #8: DDK kinase regulatory subunit DBF4
+Macromolecule #9: DNA (53-MER)
+Macromolecule #10: DNA (53-MER)
+Macromolecule #11: ADENOSINE-5'-TRIPHOSPHATE
+Macromolecule #12: MAGNESIUM ION
+Macromolecule #13: ZINC ION
+Macromolecule #14: ADENOSINE-5'-DIPHOSPHATE
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.6 |
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Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: blotted for 3 seconds before plunging. |
Details | The entire MCM loading and phosphorylation reaction was applied to the EM grid. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 18135 / Average exposure time: 9.0 sec. / Average electron dose: 51.3 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.1 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 130000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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Refinement | Protocol: FLEXIBLE FIT | ||||||||||||
Output model | PDB-7p5z: |